Methyl pyrrolidinone chitosan, production process and uses thereof

ABSTRACT

Modified chitins and chitosans with recurring units of the formula ##STR1## wherein R denotes --NH 2 , --NHCOCH 3  and R&#39;, with R&#39; being present in at least 30% of the recurring units, characterized in that R&#39; represents a group of the formula ##STR2## in a proportion of at least 90%. Due to their favourable biological properties, these materials are advantageously useful in medical and cosmetical applications.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel class ofwater-soluble, modified polysaccharides derived from chitosans, hereindicated as 5-methylpyrrolidinone chitosans, where the glucan chainscarry, at position 2,5-methylpyrrolidinone pendant groups. An essentialcharacteristic property of 5-methylpyrrolidinone chitosans is their highsusceptibility to depolymerization by lyozyze. When5-methylpyrrolidinone chitosans are applied in vivo to a wound, thischaracteristic provides an efficient way to stimulate macrophages andspleen cells, and to favor the ordered deposition of collagen, whileproviding glucosamine and N-acetylglucosamine monomers no thebiosynthetic route of hyaluronic acid and glycosaminoglycans. Thisexceptionally favorable biochemical significance enables5-methylpyrrolidinone chitosans to heal wounds in connective tissuesotherwise difficult or impossible to heal, such as bone and meniscalcartilage. Thus, a further object of the present invention is to providemedical items made of 5-methylpyrrolidinone chitosans, exertingexcellent therapeutic effects on human tissues.

Due to the mentioned characteristics, 5-methylpyrrolidinone chitosansare exceptionally useful in cosmetical applications as well. Thus, afurther object of the present invention is to provide cosmetical itemsmade of or containing 5-methylpyrrolidinone chitosans.

BACKGROUND OF THE PRESENT INVENTION

The current use of wound dressing materials is based mostly on empiricalknowledge rather than on real scientific understanding of the healingprocess. A certain number of characteristic properties of materialssuitable for wound dressing has been clarified. They are: capacity tofacilitate removal of exudates and toxic compounds, capacity to maintainhumidity at the interface wound tissue/dressing, capacity to permit gasexchange and thermal insulation, protection against secondaryinfections, easy removal from the wound without damaging the newlyformed tissue.

In the course of the last few years, polysaccharide based materials havebeen made available for wound treatment and general medications; theypossess most of the characteristics indicated above while retainingspecific properties. Important commercial products are: cross-linkeddextran (Debrisan®, Pharmacia), polyacrylamid agar (Geliperm®,Geistlich), carboxymethyl cellulose (Comfeel®), hyaluronic acid(Connettivina®, Fidia). Very little is known at the histological levelabout the effects of these polysaccharide based remedies, whilst theclinical data are abundant and the physico-chemical informations arewell known. For no one of these dressings, however, a real biologicalsignificance is shown. As far as chitin-based wound dressings areconcerned, in Japan one product is commercially available (Beschitin®,Unitika), which is a non-woven fabric manufactured from chitinfilaments. So far, no commercial exploitation, has been made ofchitosan-based medical items and very limited research has been done insuch field.

Previous research on chitin-based wound dressings

Balassa [DE 1,906,155 and DE 1,906,159 (1969); GB 1,252,373 (1971)]showed that pulverized chitin aids wound healing based on observationsrelating to mechanical resistance of the scar tissue. The same author[U.S. Pat. No. 3,632,754 (1972)] claimed "a process for facilitatingwound healing" indicating chitin as "a wound healing accelerator" [alsoU.S. Pat. No. 3,914,413 (1975)]. In a previous work [Am. J. Surgery,119, 560-564 (1970)] he indicated that chit in is "physiologicallysoluble" as a consequence of the effect of lysozyme; even in the absenceof histological data, it was suggested that N-acetylglucosamine isimportant for the orientation and cross-linking of collagen, and thaturidine diphosphate N-acetylglucosamine is a key compound in thebiosynthesis of hyaluronic acid.

Widra [EP 0,089,152 (1983)] and Miyata et al. [Jpn. Kokai Tokkyo Koho JP86,141,373 (1986)] described associations of chitosan and keratin orcollagen to be applied to a wound in the form of films. Scope of theirinventions was to provide medical items corresponding to above listedcriteria; they did not however discover any biological or histologicaleffect of chitosan on tissue components.

Yano et al. [Mie Med. J., 35, 53-56 (1985)] cast doubt on the presumedaction of chitin on larger collagen production and demonstrated thatchitin increases traction resistance compared to controls, but did notincrease the collagen quantity in the healing tissues.

Ohshima et al. [Eur. J. Plastic Surg., 10, 66-69 (1987)] reported thatwound dressing made of chitin fibers obtained by spinning organicsolutions were used on 91 patients and found to be very satisfactory interms of pain alleviation, adhesion to wound and removal of fluids.Similarly [Jpn. Kokai Tokkyo Koho JP 57,143,508 (1981) ], gauzes made ofchitin fibers were proposed, for which, however, the physicalcharacteristics only were provided. According to the same applicant[Jpn. Kokai Tokkyo Koho JP 82,11,258 (1982)] they are also useful asbinders of fibers of different nature.

Malette and Quigley [U.S. Pat. No. 4,532,134 (1985)] used chitosanacetate salt to heal wounds. They speculated that if fibrin clotformation is avoided, fibroblasts would not be stimulated and cellscould replicate the lost tissue and reduce thickness of scar tissue.They also observed reduced callus formation in the healing of bone indogs.

It is apparent from the above literature, as well as from the study ofpertinent scientific evidence so far produced, that modified chitosanshave not yet been considered for the medication of wounds, burns andother affections, in the various possible forms such as gauzes,membranes, films, non-woven fabrics, pads, fleeces, gels, not only madeof a modified chitosan alone, but not even containing an association ofa modified chitosan and other suitable materials. It is also apparentthat modified chitosans have never been used on patients.

The prior art, therefore, does not disclose how to improve thebiodegradability of amorphous chitin administered to the human body, norhow to heal certain connective tissues, such as bone and meniscalcartilage whose treatment has challenged not only chitins and chitosansbut also most of the remedies so far proposed. The prior art does notdisclose how to heal wounded or infected tissues in aged patientssuffering also from systemic diseases limiting their capacity to heal awound.

An explanation of the absence of studies on the use of modifiedchitosans on the human body is the insolubility of modified chitosans inthe physiological pH range, and the absence of gel forming ability inmost of them, these factors being a new kind of limitation against theiruse in wound dressing. For instance, N-carboxymethyl chitosan andglutamate glucan from crab chitin are insoluble [Muzzarelli and Zattoni,Intl. J. Biol. Macromol., 8, 137-143 (1986); Muzzarelli, U.S. Pat. No.4,835,265 (1986)], as well as other modified chitosans in the form ofmonosubstituted amides carrying carboxyl functions and obtained fromorganic anhydrides [Muzzarelli et al., Chitin in Nature and Technology,Plenum, New York, 1986]; the same holds for non-functionalized amidesuch as N-stearoyl and N-decanoyl chitosans [Hirano and Tokura, Chitinand Chitosan, p. 71, Jpn. Soc. Chitin, Saporo, 1982]. Chitosanderivatives carrying sugar moieties still yield gels, due to chainassociations, mostly hydrogen bond formation [Yalpani et al.,Macromolecules, 17, 272-281 (1984)]. All of the N-alkyl chitosansstudied (secondary amines) are gels [Muzzarelli et al., J. Membr. Sci.,16, 295-308 (1983)], and N-alkylidene chitosans (6 to 12 carbon atoms)are insoluble [Kurita et al., Intl. J. Biol. Macromol., 10, 124-125(1988)].

The only water-soluble modified chitosan so far reported isN-carboxybutyl chitosan, obtained from chitosan and levulinic acid[Muzzarelli et al., Carbohydr. Polymers, 11, 307-320 (1989)]; thoseauthors, however, did not teach the application of N-carboxybutylchitosan to the human body for medical purposes.

On the other hand, the introduction of certain novel functions intochitosan deprives chitosan of certain desirable characteristics; forexample, inorganic esters of chitosan, such as chitosan sulfate estersare deprived of film-forming capacity and bacteriostatic capacity(Muzzarelli et al., Carbohyr. Res., 126, 225-231 (1984)], which are wellassessed characteristic properties of plain chitosan and which aredesirable properties of a modified chitosan to be used as a medicalmaterial.

Aspects indicating that the pyrrolidinone function is desirable inmedical items

Aspects which make the presence of the pyrrolidinone function desirablein medical materials are the following, and arise mainly forminformation on poly(vinylpyrrolidone), a widely used product.

a) Pyrrolidone has the same ring of proline and hydroxyproline which aremonomeric unit of gelatin; in polymers it is deprived of the hydrogenatom on the ring nitrogen. Monomeric units carrying a pyrrolidone moietyare therefore unable to form hydrogen links, and pyrrolidone polymersshould be superior to gelatin in behavior. Poly(vinylpyrrolidone) existsin fact as a viscous solution (not a gel) and imparts hydrophilicity.Lack of hydrogen bonds permits to expose no water the ═N--C(R)═Ofunctions acting on the solvent capacity of water itself [Ling, InSearch of the Physical Basis of Life, p. 176, Plenum Press, New York,1984].

b) It is known that poly(vinylpyrrolidone), a non-ionic polymer, doesnot produce inflammation when applied to the cornea in the rabbit and itis biocompatible [Gebelein and Carraher, Bioactive Polymeric Systems, p.24, Plenum Press, New York, 1985].

c) The poly(vinylpyrrolidone) is a filmogenic substance used toreinforce membranes, either as a mixture or as a copolymer [Gebelein andCarraher, Bioactive Polymeric Systems, p. 144, Plenum Press, New York,1985].

d) A coating made of poly(vinylpyrrolidone) imparts biocompatibility,thus poly (vinylpyrrolidone) has been tried on patients in associationswith various drugs [Chiellini and Giusti, Polymers in Medicine, p. 188,Plenum Press, New York, 1983].

e) Water-alcohol mixtures are solvents for poly(vinylpyrrolidone), to beused as a binder of pigments in liners, mascara, lipsticks and othercosmetics, besides shampoos. It is also an ingredient of sprays for haircare. Pyroglutamic acid and its salts and esters, which contain a

pyrrolidone ring, are widely used in cosmetics [Proserpio, Eccipienti,Sinerga, Milano, 1985].

The prior art, however, does not disclose the introduction ofpyrrolidinone or substituted pyrrolidinone ring into a polysaccharide asa covalently bound side-chain, and the use of such products in medicaland cosmetic applications.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

It is, therefore, an object of the present invention to provide modifiedchitins and chitosans with recurring units of the formula ##STR3##wherein R denotes --NH₂, --NHCOCH₃ and R' with R', being present in atleast 30% of the recurring units, characterized in that R' represents agroup of the formula ##STR4## in a proportion of at least 90%.

Furthermore, it is an object of the present invention to provide achemical process for the production of the modified chitins andchitosans as characterized above.

Finally, it is an object of the present invention to provide medical andcosmetical items made of or containing the modified chitins andchitosans as characterized above.

The advantageous aspects of the present invention are essentially basedon:

1) a novel synthetic route to modified chitosans carrying5-methylpyrrolidinone moieties, named 5-methylpyrrolidinone chitosans;

2) high suspectibility of 5-methylpyrrolidinone chitosans to thehydrolytic action of lysozyme, leading to enhanced biologicalsignificance and effects when applied to wounded parts of the human bodyfor medical purposes;

3) enhanced functional effects when applied to healthy parts of thehuman body for cosmetic purposes.

Chemical aspects of 5-methylpyrrolidinone chitosans

According to the present invention, it was surprisingly found that thereaction between chitosan and levulinic acid can be conducted in such away as to form N-substituted anhydroglucosidic units where the nitrogenatom is in common to both the glucosamine and the 5-methylpyrrolidinonechitosan moieties. This can be achieved by adopting experimentalconditions drastically different than those indicated by previous art,namely much higher chitosan concentration, typically 20 g/l for highmolecular weight chitosans, 50 g/l for medium molecular weightchitosans; higher pH values during the hydrogenation step, typically5.6; higher atomic hydrogen concentration, typically 70 g/l of sodiumborohydride, or by performing catalytic hydrogenation with hydrogen gas;prolonged hydrogenation time, at least 3 hours. The reaction temperaturemay be chosen between 20 and 60° C., preferably at about 25° C. Thanksto the hydrogen mobility on the nitrogen atom, justified by thetautomery between ketimine and enamine under suitable experimentalconditions, the carboxyl group eliminates water thus yielding a stablelactam which lends itself to facile hydrogenation in the alpha-betapositions (see reaction scheme 1). The reaction may be carried out underessentially the same conditions but employing pseudo levulinic acid,angelica lactone or levulinate esters (see reaction scheme 2) asstarting materials. When employing levulinate esters which are unable todissolve the chitosan powders, the latter is preliminarily dissolved inan organic acid, such as, preferably, acetic acid. ##STR5##

The modification of chitosan to disubstituted amide is a novel aspect ofthe present invention, especially when levulinic acid is used for such apurpose. A further aspect of novelty of the present invention is theproper simultaneous combination of the experimental conditions neededfor the production of 5-methylpyrrolidinone chitosans. Whilst theliterature concerning levulinic acid reports the capacity of levulinicacid to produce cyclic compounds identified as lactams, with formationof the pyrrolidone ring [R. H. Leonard, Ind. Engin. Chem., 48, 1330-1341(1956); M. Kitano et al., Chem. Econ., Engin. Rev., 7, 25-29 (1975); W.L. Shilling, Tapppi, 47, 105A-108A (1965)], such publications did not,however, indicate the possibility of lactam ring formation by levulinicacid reacted with chitosans, such a possibility, though hypothesized,having technically and experimentally escaped to many authors fordecades. The formation of the 5-methylpyrrolidinone moiety in themodified chitosan, achieved in the context of the present invention byadopting an unusual see of experimental conditions, is an novelalternative to the formation of the N-carboxybutyl group [R. Muzzarelliet al., Carbohydr. Polymers 11, 307-320 (1989); R. Muzzarelli, U.S. Pat.No. 4,835,265 (1986)]. In fact, such prior art produces N-carboxybutylchitosan and demonstrates its identity, without providing anysuggestions for the alternative production of 5-methylpyrrolidinonechitosan. Although the first of the aforementioned publication indicatedthat part of the N-carboxybutyl function was believed to exist in the5-methylpyrrolidinone form, exact analytical investigations meanwhileshowed that this form, due to the reaction conditions of the synthesis,was generated only in a very low proportion, i.e. in less than 10%.N-Carboxybutyl chitosan, as described in the aforementioned prior art,was obtained at low chitosan concentration (6 g/l) and at a low pH value(4.0), moreover, the hydrogenation step was short and the reducing agentwas mild (sodium cyanoborohydride). These conditions are very far fromthose which allow lactam formation, and possibly do not even permit fullsubstitution, i.e. less than 30%, a major proportion of the repeatingunits of the N-carboxybutyl chitosan still being in the free amine form.

On the opposite, the conditions adopted in the present process do notonly lead to formation of cyclic (lactam) moieties, but also to higherdegrees of substitution, i.e. at least 30%. Therefore, according to thepresent invention, modified chitins and chitosans are obtained where thecarbon-2 atoms in the recurring units carry --NH₂, --NHCOCH₃ and R',with R' being present in at least 30% of the recurring units,characterized in that R' represents a group of 5-methyl-2-pyrrolidone ina proportion of at least 90%. The hereby reported 5-methylpyrrolidinonechitosan is an unique and novel macromolecular compound which should notbe confused with the chitosan pyroglutamate salt (Kytamer®, Amerchol)the latter being simply a mixture of chitosan (a macromolecule) andpyroglutamic acid (independent monomers).

The instrumental evidence testifying the identity of5-methylpyrrolidinone chitosan is the following:

a) Behavior towards metal ions

When 5-methylpyrrolidinone chitosan is titrated with sodium hydroxide orpotassium hydroxide, just 9% of the stoichiometric quantity of alkalimetal ion, referred to theoretical carboxyl group, combines with the5-methylpyrrolidinone chitosan. This fact demonstrates that the carboxylgroup exists as such [--COOH] to a very limited extent. Being unable toform sodium and potassium salts, the carboxyl group appears to beengaged in lactam ring formation. As a confirmation, the alkalimetrictitration according to Broussignac gives an amine titer much lower thanthe one corresponding to the chitosan used. As for transition metalions, while N-carboxybutyl chitosan exerts chelating action towardscopper and lead, 5-methylpyrrolidinone chitosan shows a limited and aspecific complexing ability towards copper alone.

b) Proton nuclear magnetic resonance spectrometry

Authentic methyl pyrrolidinone shows two twin signals at 1.113 and 1.144ppm in the spectrum, besides signals at 2.2 and 3.7; said signals do notappear in the spectrum for authentic levulinic acid which is flat in theregion 0-2 ppm and has one signal at 2.176 ppm followed by two sets oftriplets centered an ca. 2.55 and 2.80 ppm. In the spectrum forN-carboxybutyl chitosan the signals corresponding to those of levulinicacid can be found in addition to the broad signal for chitosan, whilstin 5-methylpyrrolidinone chitosan the signals corresponding to themethyl pyrrolidinone characterize the spectrum and are accompanied bylower signals for levulinic acid, which evidently form a salt with theremaining unmodified primary amine. This is in agreement with datapublished on levulinic acid [Sunjic et al., Kern. Ind. (Zagreb), 33,599-602 (1984)].

c) ¹³ C Nuclear magnetic resonance spectrometry

These spectra for 5-methylpyrrolidione chitosan reveal signals in theinterval 20-40 ppm assigned to methyl and methylene groups newlyintroduced with levulinic acid, in agreement with data reported forpoly(vinylpyrrolidone) [D. A. Brant, Solution Properties ofPolysaccharides, p. 127, ACS, Washington, 1981] and those reported forauthentic levulinic acid [V. Sunjic et al., Kem. Ind. (Zagreb), 33,599-602 (1984)]. The signal for the carboxyl group is hardly visible,its height being less than twice the background noise. ForN-carboxybutyl chitosan, on the other hand, the carboxyl group givesharp signals at 174 and 182 ppm. Different ratios between the methylenesignals also reveal a different chemical environment in the linear sidechain.

d) Fourier transform infrared spectrometry

For 5-methylpyrrolidione chitosan samples examined in the form of a thinfilm prepared at pH 6, bands at 1400 cm⁻¹ for methylene and methylgroups, at 1690 for the lactam and at 1700 for the amide carbonyl revealthe altered structure of the polysaccharide; moreover, the free amineband at 1590 is depressed [for assignments, A. Wochowics et al., ActaPolym. (Varsavia), 38, 194-189 (1987)]. A comparison of this spectrumwith the one for N-carboxymethyl chitosan, which certainly is exempt ofcyclic side structures [R. Muzzarelli et al., Carbohydr. Res., 107,199-214 (1982), FIG. 3] showed that the major difference is certainly inthe region 1500-1600 cm⁻¹, indicative of the prominence of the lactamband for 5-methylpyrrolidionone chitosan. Also, for protonated thinfilms of 5-methylpyrrolidione chitosan, the absence of the 1730 cm⁻¹band indicated absence of protonated carboxyl groups.

e) Biodegradability by lysozyme

The hydrolytic depolymerization of 5-methylpyrrolidinone chitosan by henegg white lysozyme was studied in vitro. Viscometric measurements atthree temperatures (25°, 37° and 50° C.) and five 5-methylpyrrolidinonechitosan concentrations (from 9 to 27.3 g/l), provided kinetic dataincluding the Michaelis constant, 1×10⁻⁴ mmol/l. Linearity was observedwhen log K_(M) was plotted versus 1/T. The viscosity decrease over 50min period was a linear function of temperature, independent of initialsubstrate concentration. Therefore, 5-methylpyrrolidinone chitosan ishighly susceptible to the hydrolytic action of lysozyme and its initialhigh average molecular weight (ca. 700×10³ dalton) was decreased down tovalues close to 10×10³ dalton. In comparison with chitosan, it wassurprisingly found, according to the present invention, that5-methylpyrrolidinone chitosan was much more sensitive to the lysozymeaction. In fact, chitosans with average molecular weights in the range166-191×10³ were degraded by lysozyme down to values in the range19-79×10³ after 30 hours [Yomota et al., Yakugaku Zasshi, 110, 442-446(1990)] and the decrease of viscosity in 50 min was just in the range30-35% for chitosans of similar degree of deacetylation. Fullydeacetylated chitosans were found not to be hydrolyzed by lysozyme atphysiological pH values. The uniquely high susceptibility of5-methylpyrrolidinone chitosan to depolymerization by lysozyme, heredescribed for the first time, is a key factor for the surprisingfavorable results in wound healing.

Biological significance

The most important and surprising biological activities exerted by5-methylpyrrolidinone chitosan on human tissues, according to thepresent invention, here described for the first time, are the following,among others: healing of wounded meniscal tissues, healing of decubitusulcers, depression of capsule formation around prostheses, limitation ofscar formation and retraction during healing, and osteoconduction. Foreach of these topics, details are given below in the Examples section.

The rationale for the use of 5-methylpyrrolidinone chitosan is mainlybased on its surprisingly high and unmatched susceptibility to lysozymehydrolytic action. Once applied to a wound, 5-methylpyrrolidinonechitosan becomes immediately available in the form of oligomers producedunder the action of lysozyme, for the following actions:

a) stimulation of macrophages and spleen cells;

b) providing aminosugars for incorporation into glycosaminoglycans ofnewly formed connective tissues;

c) favouring diffusion of factors and other compounds, cellproliferation and epithelial cell migration, in view of the gel formingability of 5-methylpyrrolidinone chitosan in the boundary regions incontact with the wound tissues,

d) preventing regression to a vascular scar tissue.

As far as macrophage activation by oligomers generated from5-methylpyrrolidinone chitosan is concerned, activation means productionof interleukin-1 which promotes fibroblast proliferation, leading toordered collagen deposition. The teaching from cited prior art [Maletteand Quigley] appears to be misleading in this report.

Activated macrophages also secrete interferon and tumor necrosis factorwhich provide a favourable situation for the further production ofN-acetylglucosaminidase isoenzymes capable to hydrolyze the abovementioned oligomers to monomers. The latter become available forincorporation into hyaluronic acid, keratan sulfate and chondroitinsulfate via the well known biosynthetic route of these macromolecules.Thus, on one hand, 5-methylpyrrolidinone chitosan favours orderedcollagen fibril formation rather than disordered scar tissue deposition,which is an essential condition for imparting functionality to therepair tissue; and on the other hand, creates the biochemical conditionsfor the availability of aminosugars, i.e., building blocks of thepolysaccharides present in the extracellular matrix. These two combinedactions, which are unique to 5-methylpyrrolidinone chitosan, accordingto the present invention, explain the efficacy and the usefulness of5-methylpyrrolidinone chitosan in wound treatment. The presence of saidcompounds in the wound site also explains the angiogenetic action andabsence of inflammation.

According to the favourable properties of 5-methylpyrrolidinonechitosan, this material may be advantageously used in wound dressingmaterials, e.g. in the form of freeze-dried materials, powders, films,non-woven fabrics, adhesive tapes, bandages, membranes, solutions,xerogels, hydrogels, filaments, textiles, tissues, lotions, creams. Suchwound dressings are intended for internal and external use, to healburns from heat and solar radiation, surgical and traumatic wounds,infections and decubitus ulcers.

Due to the same reasons, it may be used as well as cosmetic ingredient,e.g. in the form of freeze-dried materials, powders, films, membranes,solutions and gels. These cosmetic ingredients are suitable as such, toform facial masks or to be used as cosmetic applicators, or are to beformulated into emulsions, shampoos, hair-care preparations, gels,liquid crystal compositions, liposomes, mainly to confer functionalproperties to the final preparations, or more simply to modify theirrheology or stabilize certain systems such as liposomes andmicroemulsions.

Furthermore, it may be applied in coatings, e.g. for prosthetic andorthopaedic devices, manufactured with silicones, polyurethanes,hydroxyapatites and other biomaterials. These coatings are mainlyintended to confer biocompatibility to the medical items, and to avoidtissue reactions when the latter are implanted.

Finally, it may be applied in supports for delayed release of drugs,e.g. in the form of freeze-dried materials, powders, films, membranes,solutions, gels, filaments, textiles, tissues, lotions, creams, tablets.These excipients for delayed release are mainly intended to enhancewater-solubility of poorly soluble drugs and to make such drug availableto the organism over an extended period of time after administration.

Advantages of 5-methylpyrrolidinone chitosan over N-carboxybutylchitosan are the following: 5-methylpyrrolidinone chitosan beingdeprived of free carboxyl groups has no amphoteric behavior asN-carboxybutyl chitosan has, and therefore is more cationic in characterwhich leads to better antimicrobial action; the lactam rings in5-methylpyrrolidinone chitosan are capable to prevent hydrogen bondassociation and therefore 5-methylpyrrolidinone chitosan is also solubleat alkaline pH values whereas N-carboxybutyl chitosan is not; in view ofthe stability of the lactam ring, 5-methylpyrrolidinone chitosan is morestable in the course of time.

The chemical and technological processes, and the clinical andhistological effects which constitute the embodiment of the presentinvention are given in the following examples, which, however, are notintended to be a limitation of the applicability of this invention.

EXAMPLES Example 1--Preparation of 5-methylpyrrolidinone chitosan

Crustacean chitosan powder (9 g) was suspended in water (500 g) and keptunder stirring for at least 1 hour. Levulinic acid (9.7 g) was thenpoured into the reaction vessel, and pH became in general 4.3, which isconvenient for the Schiff reaction to take place, at 25° C. Sodiumborohydrided solution (20 ml, 70 g/l) was delivered over a time periodof at least 3 hours, to reach pH 5.6. The resulting solution wasdialysed against distilled water for 3 days (3 changes) and used forpreparations according to Example 2. In case of medium molecular weightchitosans (50-100×10³ dalton), initial concentration was higher thanindicated above, typically 50 g/l. Chitosan concentration, pH values andhydrogenation time, as indicated in this Example, are crucial parametersfor the satisfactory preparation of 5-methylpyrrolidinone chitosans. Aportion of the product was submitted to analysis according to the aboveindicated criteria and methods, by infrared spectrometry and nuclearmagnetic resonance spectrometry, and found to correspond to thestructure of 5-methylpyrrolidinone chitosan.

Example 2--Absence of chelating ability towards lead

Absorption spectra in the ultraviolet region were recorded with the aidof a Varian spectrophotometer by the twin-cells method which permits thechelate spectrum to be recorded with no interference from otherabsorbing species. This was done on lead perchlorate solutions havingconcentration 0.06 mol/l in admixture with either 5-methylpyrrolidinonechitosan or N-carboxybutyl chitosan (6 mmol/l). In the latter case,absorption bands centered at 227 nm were recorded and their height wasproportional to the molar ratio between lead ion and sugar unit; on theopposite, in the case of 5-methylpyrrolidinone chitosan, a flat spectrumdeprived of any absorption band was recorded, thus revealing thedifferent behavior towards metal ions, due to the presence of lactam in5-methylpyrrolidinone chitosan.

Example 3--Preparation of wound dressings

Aqueous solutions containing 5-methylpyrrolidinone chitosan can bediluted with alcohols, including ethanol and 2-propanol. They arecompatible with other polymer solutions including gelatin,poly(vinylalcohol), poly(vinylpyrrolidone) and hyaluronic acid. For thepreferred preparation of wound dressing materials, aqueous solutions atpH values close to neutrality, preferably 6, after dialysis, werefreeze-dried to yield fleeces. The latter were optionally laminatedbetween stainless steel plates to reduce their thickness to ca. 1-2 mm,and after sealing them in double plastic envelopes, were submitted to ⁶⁰cobalt gamma-ray irradiation at 1.4 Mrad. The resulting soft and sterilematerial could be applied on any surgical or traumatic wound. As analternative to lyophilization, thermal drying was used: the5-methylpyrrolidinone chitosan solutions were evaporated preferably at50° C. on plastic or glass plates. Both procedures were used to coatorthopaedic objects.

Example 4--Uses in plastic surgery

In patients undergoing plastic surgery, donor sites on the front side ofthe right leg were treated with fleeces of freeze-dried5-methylpyrrolidinone chitosan to promote ordered tissue regeneration.Compared to controls (left leg of the same patient), betterhistoarchitectural order, better vascularization and absence ofinflammatory cells were observed at the dermal level, while feweraspects of proliferation of the malpighian layer were reported at theepidermal level.

In the late stages of the normal process of the wound healing, whencollagen synthesis declines and high oxygen tension is no longerrequired, many new vascular channels regress: the wound becomes usuallyavascular and undergoes transformation into a scar with limited tissueelasticity. Regression of angiogenesis took place in all cases as soonas 5-methylpyrrolidinone chitosan was no longer administered or had beenabsorbed; nevertheless, the resulting connective tissue appeared orderlystructured and endowed of good functionality. The 5-methylpyrrolidinonechitosan provided a tridimensional supporting lattice favouring theepithelial cell migration, and in any way, modulatedre-epithelialization.

Example 5--Use in plastic surgery with insertion of expanders

An aspect of importance in plastic surgery is capsule formation around aforeign body. Anomalous deposition of connective tissue takes place whenthe dynamic equilibrium between synthesis and breakdown is altered,leading to fibrosis, an ubiquitous, aspecific and disordered increase ofcollagen. The fibrous capsule formed after implanting a tissue expanderunder the skin is a macroscopic aspect of such reparative process. Theconnective tissue cellular components responsible for the organizationof the collagen lattice, determine such a structure by exerting orientedfraction forces. Steps leading to capsular structure are:

1) mesenchymal elements are attracted and concentrated were theattraction is stronger and increase it;

2) fibroblasts aligned along the major axis of the extracellular fiberbundles tend to orientate the fiber along the axis, thus amplifying theprocess of structural orientation.

Silicone expanders coated with 5-methylpyrrolidinone chitosan wereinserted into surgical wounds, and the formation of capsular tissue wasstudied by electron microscopy. During all of the stems of capsularorganization, 5-methylpyrrolidinone chitosan sustained correctproliferation and organization, and stimulated physiologically thetissue repair process; angiogenesis was favoured while fibrogenesis wasdepressed.

The formation of vascularized connective tissue with copious mesenchymalelements and reduced collagen components indicated the ability of5-methylpyrrolidinone chitosan to assist newly formed tissue inretaining good trophicity and loose state, which are favourablephysiological characteristics. 5-methylpyrrolidinone chitosan increasedthe interfibrillar amorphous substance in the dermal region close to theexpander, and reduced the damage generated by the foreign body. Theloose capsular tissue formed in its presence was less prone tocontraction-retraction, during maturation.

Example 6--Use in orthopedics

The well-known difficulties in obtaining spontaneous repair of themeniscal structure are a real challenge to the use of biomaterialsintended for promotion of a guided repair of the carthilagineous tissue.

The angiogenetic properties of 5-methylpyrrolidinone chitosan assumedparticular importance in the repair process of the meniscus. In fact,such repair is conditioned by the presence of vessels that5-methylpyrrolidinone chitosan could be able to extend from adjacentcapsular structure. Results indicated that 5-methylpyrrolidinonechitosan was well tolerated at the articular-synovial level. It alsofavoured and stimulated the repair processes which do not take placespontaneously in the meniscus.

In the meniscal areas close to the synovial lining, the angiogeneticstimulus provided by 5-methylpyrrolidinone chitosan led to furtherrepair processes of the meniscal tissue as indicated by morphologicaldata. Repair did not take place when the surgical lesion was excessivelydistant from the vascular structure of the meniscus-synovium junction.Observations made on the synovium 45 days after the application of5-methylpyrrolidinone chitosan, showed that the synovial membrane hadcells layered on a subintimal stromal tissue exhibiting tightly packedcollagen fibers among which mesenchymal cells and vascular structureswere visible. After the same number of days, the meniscal tissue wascharacterized by structural reparative aspects with irregularlydistributed collagen bundles, evolving toward cartilagineous tissue;microvessels were also present. In conclusion, 5-methylpyrrolidinonechitosan was found suitable for healing meniscal lesions.

Example 7--Use in dental surgery

Osteoinduction is a phenomenon leading to growth of capillaries,perivascular tissue and bone-generating cells, proceeding from thebottom of the alveolar bed and invading most of the space occupied by5-methylpyrrolidinone chitosan. Surgical wounds produced in order toremove the apical part of an infected tooth, or to remove in toto awisdom tooth were filled with freeze-dried 5-methylpyrrolidinonechitosan. At the x-ray examination, one month after surgery, theformation of native bone was evident in all the 6 patients treated.Biopsies taken on 2 patients 4 months after surgery, and examined at theelectron microscope, confirmed the generation of bone tissue in thealveolar region, which filled the space occupied by5-methylpyrrolidinone chitosan. As a consequence, functionality of thesame tooth (apicectomy) and of the adjacent teeth (extraction of wisdomtooth) was much improved, in comparison to control patients treated intraditional way. In no case inflammation took place to extents exceedingthe normal inflammation for controls.

Example 8--Use in Gerontology

In aging skin, collagen undergoes cross-linking reactions andphysico-chemical alterations; proteoglycans decrease in general andtheir percent ratios are altered. Vascular walls undergo thicknessincrease and a reduced quantity of oxygen reaches the cutaneous tissuewith unfavourable consequences in case of ulcers and burns.Hypertension, oedema, atherosclerosis and diabetes further reduce thequantity of oxygen, nutrients and cells having defensive action(leukocytes and macrophages) reaching the cutaneous tissue.

Patients (10, average age 62) suffering from leg ulcers were submittedto treatment with 5-methylpyrrolidinone chitosan fleeces or gels, forperiods of time up to 30 days. Compared to controls, a more rapidepithelialization was remarked (7 days instead of 15-20 days). In nocase infections occurred and good hemostasis was observed. From themorphological point of view, controls showed the usual disordereddeposition of collagen fibers, whilst in patients treated with5-methylpyrrolidinone chitosan, a correct histoarchitecture of theregenerated skin was observed, in particular an ordered organization andvascularization of the derma.

Example 9--Preparation of cosmetic facial masks

Facial masks were prepared by pouring a solution (400 g, 16 g/l) offreshly prepared 6-methylpyrrolidinone chitosan on a flat polystyrene orglass surface (20×30 cm) and drying at 50° C. in a ventilated oven for12 hours. The 5-methylpyrrolidinone chitosan masks were also preparedwith the addition of ethoxylated castor oil as a plasticizer (5%). Thetransparent films thus obtained had the peculiar characteristic offorming a gel when contacted with water. Therefore, when contacted withmoisturized facial skin, the facial masks partially gelified on thecontact side and fully adhered to the skin. A group of persons (5ladies) participating in the evaluation of these masks reportedunanimously that the application was comfortable and pleasant, due tocomplete and durable adhesion, agreeable refreshing feeling andattractive transparency. After removal, effective skin cleaning and adurable moisturizing effect were reported. Advantages over collagenmasks were: transparency, full adhesion, gel-forming ability andlong-lasting effect.

Example 10--Preparation of cosmetic creams

Oil-in-water emulsions were prepared to contain: Xalifin-15®[ethoxylated fatty acids C₁₂₋₁₈ ], 4.5 g; Glucamate SS20®[methylclucoside stearate, ethoxylated], 5.4 g; Glucate SS®[methylglucoside stearate], 3.6 g; Cetyl alcohol [n-esadecanol], 13.5 g;Cetiol® [oleyl oleate], 45.0 g; Karite butter, 9.0 g; Water, 315 g;Gram-1® [imidazolinyl urea], 1.25 g; Kathon-CG® [isothiazolinonechloride], 0.75 g. This formulation was taken as a reference. Byreplacing a portion of water (125 g) with as much 5-methylpyrrolidinonechitosan 1% solution, a cream was obtained with superior organolepticcharacteristics, filmogenicitiy, hydrating capacity and skin-protectiveaction. The rheological behavior were typical for plastic systems. Thepresence of 5-methylpyrrolidinone chitosan did not alter the sensitivityof the cream no the deformation, but promoted restructuring aftermechanical stress. Ultraviolet irradiation yielded very modest chromaticvariations.

I claim:
 1. A modified chitin and/or chitosan polymer consistingessentially of monomeric units of formula I ##STR6## wherein R is --NH₂,--NHCOCH₃ or R', whereinR' is an open chain N-carboxybutyl group or theisomeric cyclic 5-methylpyrrolidinone group of formula II ##STR7##wherein 90% of R' is present as the 5-methylpyrrolidinone of formula II;and wherein said monomeric units of formula I wherein R is R' compriseat least 30% of the total monomeric units in the modified chitin and/orchitosan polymer.
 2. A wound dressing material made in whole or in partwith a polymer of claim 1, in the form of a freeze-dried material,powder, film, non-woven fabric, adhesive tape, bandage, membrane,solution, xerogel, hydrogel, filament, textile, tissue, lotion or cream.3. A support for delayed release of a drug made in whole or in part witha polymer of claim 1, in the form of a freeze-dried material, powder,film, membrane, solution, gel, filament, textile, tissue, lotion, creamor tablet.
 4. A cosmetic ingredient made in whole or in part with apolymer of claim 1, in the form of a freeze-dried material, powder,film, membrane, solution or gel.